Preparation of l-leucine by fermentation

ABSTRACT

L-leucine is prepared by culturing a L-leucine-producing microorganism of the genus Corynebacterium in a culture medium containing a carbon source, a nitrogen source, inorganic material, nutrients and an isoleucine, methionine, phenylalanine or valine promoter. Mixtures of the promoters may also be employed. Corynebacterium glutamicum ATCC 21,301 and 21,335 are particularly suitable microorganisms for use in the process. Lleucine is an essential amino acid and is useful as a nutrient additive in food and feedstuffs for human and animals, respectively.

United States Patent Kurihara et al.

[ PREPARATION OF L-LEUCINE BY FERMENTATION [72] Inventors: Sumio Kurihara; Kazumi Araki; Hiroyuki Ueda; Masahiko lkumo, all of Hofu-shi,

[21] Appl.No.: 790,177

[30] Foreign Application Priority Data Jan. ll, 1968 Japan ..43/l089 [52] US. Cl.....'. ..l95/29, 195/30, 195/47, 195/49 [5 l] Int. Cl. ..C12b 1/00 [58] Field of Search .i 195/29, 28, 30, 47

[56] References Cited UNITED STATES PATENTS 3,133,868 5/1964 Takesue et a] ..l95/30 1 June 6, 1972 FOREIGN PATENTS OR APPLICATIONS 1,489,909 6/1967 France Primary Examiner-A. Louis Monacell Assistant Examiner-Robert M. Elliott Attorney-Bacon & Thomas [57] ABSTRACT L-leucine is prepared by culturing a L-leucine-producing microorganism of the genus Coryn ebacterium in a culture medium containing a carbon source, a nitrogen source, inor ganic material, nutrients and an isoleucine; methionine, phenylalanine or valine promoter. Mixtures of the promoters may also be employed. Coryne bacterium glutamicum ATCC 21,301 and 21,335 are particularly suitable microorganisms for use in the process. L-leucine is an essential amino acid and is useful as a nutrient additive in food and feedstuffs for human and animals, respectively.

7 Claims, No Drawings 1 PREPARATION OF L-LEUCINE BY mMENTATloN BACKGROUND OF INVENTION L-leucine is an essential amino acid and is useful as a nutrient additive in food and feedstufls for humans and animals, respectively.

Prior investigators have reported that L-leucinecan be obtained by using a mutant strain of Microcaccus glutamicus DETAILED DESCRIPTION OF THE INVENTION We have discovered that L-leucine can be obtained by culturing a microorganism of the genus Corynebacterium in a culture medium containing a source of carbon, asource of nitrogen, inorganic substances, various other nutrients and an isoleucine, methionine, phenylalanine or valine promoter. Mixtures of the promoters may also be employed.

As described more fully in the examples, it is possible according to this invention to easily and with sufiicient reproductivity accumulate L-leucine in an amount of more than about 10 mg/ml in the broth (sugar concentration: 10-20 g/dl), accordingly, the process of this invention is very useful for industrial production.

The mutant strains useful .in this invention have nutrient requirements which distinguish them from strains conventionally used for industrial fermentation and from the mutant strains reported in said Japanese Pat. Publication No. 14,395/1963. Particularly particularly suitable microorganisms for use in the process according to this invention are strains which are mutants of Corynebacterium glulamicum (syn. Micrococcus glutamicus disclosed in Japanese Patent Publication No. 8698/1967). Cultures of these mutants have been deposited without restriction as to their being made available to the public in the American Type Culture Collection in which they are identified as ATCC 21,301 and 21,335. Beside these strains, other similar types of mutant strains belonging to Corynebacterium glulamicum have been discovered and can also be used in the practice of the invention.

ATCC 21,301 and 21,335 are obtained from the L-glutamic acid-producing strain Cor ynebcclerium glutamicum KY-l0108 and MF-l42 respectively, by treating with N- methyl-N'-nitro-N-nitrosoguanidine.

The obtained mutant strains can be distinguished from the parent strains as follows:

When they are cultured by using minimal medium [reported in Amino Acids and Nucleic Acids, 8, 53, (1964)] in the presence of 100 y/ml of isoleucine, methionine, phenylalanine, valine or mixtures thereof at 28 C. for 30 hours,

their growths have the following properties (Table 1):

it is apparent from the table that it is possible to propagate the strains in a minimal medium without addition of amino acid but with an extremely low propagation velocity. The promoter action of the amino acid additives is readily apparent. Among the said amino acids, the promoter action of phenylalanine is clearest at the earlier stage of the propagaobtaine'd mutant strains belong to the class of strains having the relative req uiremen to the said amino acids group. The bacteriological properties of Corynebacterium glutam- 'icum are well known in light of the report in The Journal of General and Applied Microbiology,

Vol. 18, 279-301 (1967).

The culture medium which may be used for the purpose of this-invention include any and all synthetic or'natural medium containing suitable amount of carbon source, nitrogen source, inorganic substances and other nutrient.

As the carbon source in the culture medium, glucose is preferably used, but various assimilable carbon sources such as, e.g., fructose, mannose, galactose,maltose, sucrose, starch hydrolysate, molasses, glycerol, acetic acid, etc., my be used alone or in admixture.

As the nitrogen source, both organic and inorganic nitrogen compounds, such as ammonia, urea, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium salts of various other organic acids, etc., may be used.

As the inorganic substances which'may be included in the culture medium, there may be mentioned various salts of iron, manganese, magnesium, cobalt,'zinc, nickel, chromium, etc., as well as various phosphoric acid compounds.

As the amino acids which will satisfy the specific requirements' of the strains according to this invention, it is necessary to use isoleucine, methionine, phenylalanine, valine or mixtures thereof. The amount of the aminoacid to be added depends upon the culturing conditions and upon the types and nature of the amino acids to be used but it is preferred to use 50 to 1,000 'y/ml of amino acid.

As the nutrients, amino acids (e.g., cystine, cysteine, glutamic acid, etc.) and vitamins (e.g., biotin,,thiamin, etc.) can be added if desired. Yeast extract, Mieki (a soybean meal hydrolysate solution manufactured by Ajinomoto Co., Ltd. and available on the open market in Japan), com steep liquor, peptone, protein hydrolysate, mat extract, hydrolysate of microbial cells, etc., can also be'usedas nutrient-containing materials. I i

L-leucine fennentation is carried out under aerobic conditions. The culturing temperature is 25 to 40 C. and preferably 27-37 C. The pH of the medium will vary considerably but it is advantageous .to adjust the pH within the range of 5.5 8.5 with a suitable neutralizing agent (e.g., ammonia, sodium hydroxide, potassium hydroxide, calcium hydroxide, urea etc.) during cultivation. After culturing for 3-6 days, L-leucine is accumulated, but L-tyrosine may be produced as a by-product under certain culturing conditions.

After culturing, microbial cells are removed from the broth, e.g., by filtration or centrifugation, and the filtrate is treated by a suitable method, e.g., strongly acidic cation exchange resin treatment, in order to yield L-leucine. The absorbed L- leucine is eluted, concentrated and cooled in conventional manner to yield crude crystals of L-leucine. v

The following examples serve to illustrate the invention, but

are not to be considered as limiting the same.

EXAMPLE 1 A culture medium was innoculated with Corynebacterium gluramicum ATCC 21,301, the culture medium containing peptone (l gldl), meat extract (lg/d1), yeast, extract (0.5

gldl), sodium chloride (0.3 g/dl) and glucose (2 g/dl) and havlated with 1 ml of theseed culture, the preparation of which was described above. Fermentation was carried out at 28 C.

' for days with shaking. An average of 9.5 mg/ml of bleucine was accumulated the culture medium in each flask.

One literof was collected from the broths and centrifuged to remove microbial cells 'l'he filtrate was through a .resin column packed with Dianion SK No. l [strongly acidic Y cation exchange resin available from Mitsubishi Kogyo K.K., Japan] in its l-l form. The resin was washed with a 0.2M aqueous solution'of ammonia to elute L- leucine. The-fractions containing the eluted L-leucine were combined and concentrated. After standing, there were obtained crudecrystals of L-leucine (6.5 g) which were added to methanol (700 ml; 70 percent by weight). The solution was heated to dissolve the methanol and wm allowed to cool on standing. The resultant precipitate was separated and washed with methanol (70 percent by weight) to yield purified L-leucine.

I Analytical. results obtained by the use of paper chromatography and automatic amino acid analyzer demonstrated that the L-leucin'e produced by the above process had similar characteristics to L-leucine produced by the conventional process. The product obtained by the above process was biologically active and was employable for the growth of strains useful for the bioassay of L-leucine. lts melting point was 298 C. (decomposed).

EXAMPLE 2 A fermentation was carried out in a manner similar to that described in Example 1, except that a medium containing glucose (l2 g/dl), yeast extract (0.5 g/dl), ammonium sulfate (2 g/dl), KHJO (0.15 g/dl), K,HPO (0.05 g/dl), MgSO,'7H,O (0.05 'gldl), FeSO -7H,O (0.002 dd). MnSO '4H,O (0.002 g/dl), biotin (50 7/1) and CaCO; (2 g/dl) was used and the fermentation was carried out, at 29 C. for 4 days. An average of l 1.0 mg/rnl of L-leucine was'accumulated in the culture medi- EXAMPLE3 A fermentation was carried out in a manner similar to that described in Example 1, exceptthatCorynebacrerium glutamicum'ATCC 2!,335 and a medium-containingFeSO7I-I,O

tone (1 g/dl), ammonium sulfatei(-2-gldl), KHJO, (0.15 g/dl), I

K HPO (0.05gldl), MgSO.'7H; O (0.002 g/dl), biotin 7/1) and CaCO, (2 fldl), were used. An average'of'l 1.4 mg/ml-of L-leucine was flask.

, We claim: I

1. A process of preparing L-leucine, consisting essentiallyof aerobically culturing a .L-leucine-p'roducing microorganism accumulated in culture medium in each belonging to Coryneb'tmen'um gluramicum having the relative requirements to a promoter selected from the group consisting of isoleucine, methionine, phenylalanine, valine, and mixtures thereof in a culture medium 'a carbon source selected from the group consisting of glucose, fructose, mannose, galactose, maltose, sucrose, starch hydrolysate, molasses, glycerol, acetic acid "and mixtures thereof, a nitrogen source, inorganic material and nutrients and a promoter selected from the group consisting of isoleucine, methionine, phenylalanine, valine and mixtures thereof and recovering the accumulated L-leucine from said culture medium.

2. A process as claimed in claim 1 in which said microorganism is Corynebacren'unr glutamicum ATCC Z l .301 t 3. A process as claimed in claim 1 in which-said microorganism is Corynebacterium glutamicum ATCC 21,335.

4. A process a claimed in claim 1 in which the temperature 'of said culture medium is maintained within the range of 25-40'C. I v

5. A process as claimed in .claim 4 in which said temperature is within the range of2737 c.

man s a' 

2. A process as claimed in claim 1 in which said microorganism is Corynebacterium glutamicum ATCC 21,301.
 3. A process as claimed in claim 1 in which said microorganism is Corynebacterium glutamicum ATCC 21,335.
 4. A process as claimed in claim 1 in which the temperature of said culture medium is maintained within the range of 25*-40* C.
 5. A process as claimed in claim 4 in which said temperature is within the range of 27*-37* C.
 6. A process as claimed in claim 1 in which the pH of said culture medium is maintained within the range of 5.5-8.5.
 7. A process as claimed in claim 1 in which culturing is continued for a period within the range of 3-6 days. 